Ultracompetent E. coli cells?

Richard Friedman friedmrd at dunx1.ocs.drexel.edu
Tue Dec 19 18:49:20 EST 1995


In article <DJn2uq.AJ4 at ncifcrf.gov>, pnh at cockleberry.ncifcrf.gov (Paul N
Hengen) wrote:

> Andy Phillips (andy.phillips at bbsrc.ac.uk) wrote:
> 
> | Could some kind person post or email me a quick recipe for the Inoue
> | method for producing ultra-competent E.coli?
> 
> In a nutshell,
> 
> 1.  Inoculate from an overnight grown in LB
> 2.  Grow in 250 ml "SOB" at 18C until OD600 = 0.6
> 2.  On ice for 10 minutes.
> 3.  Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min.
at 4C.
> 4.  Resuspend cells gently in 80 ml of ice cold "TB".
> 5.  On ice for 10 minutes.
> 6.  Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min.
at 4C.
> 7.  Resuspend cells gently in 20 ml of ice cold "TB"
> 8.  Add DMSO to a final concentration of 7%.
> 9.  On ice for 10 minutes.
> 10. Aliquot into 1-2 ml and freeze in liquid nitrogen.
> 11. Store in liquid nitrogen.
> 
Is there any reason why they have to be stored in liquid nitrogen? If I
flas freeze in Liq N2 then store at -80oC would I loose much ?

-- 
-Rich
friedmrd at dunx1.ocs.drexel.edu
***********************************************************
* "This Genie gig has it's ups and        `(:>)~\         * 
* downs -- PHENOMENAL COSMIC POWER,              \     /  *
* itty-bitty living space."                       \___/   *
***********************************************************



More information about the Methods mailing list